Project Summary: This application will address the unmet need for superior treatment outcomes for adults with acute lymphoblastic leukemia (ALL), and will develop the tools needed for personalized treatment to allow a more expanded use of the unique anti-cancer drug called L-asparaginase. Unlike pediatric ALL, a disease with a cure rate of >90%, the cure rate of adult ALL is <40%. One significant difference between the treatment of pediatric and adult ALL patients is that only the pediatric regimen always includes the drug L-asparaginase. Indeed, it was shown that cure rates are highly dependent on using this drug, and for the patient being able to complete the full course of treatment. Unfortunately, the side effects of L-asparaginase treatment often require prematurely stopping use of this drug. These L-asparaginase side effects can be traced directly to the bacterial origin and properties of all current FDA-approved L-asparaginases (and not to the anti-cancer asparagine depletion effect of drug). Being bacterial enzymes, currently approved drugs are highly immunogenic. Although a portion of this clinical problem has recently been addressed by pegylating the enzyme, the other source of side effects, the L- glutaminase co-activity of these bacterial enzymes, still remains. We propose a strategy that would address both the immunogenic and L-glutaminase-related side effects, in which the bacterial enzymes are replaced by human-like L-asparaginases that are devoid of L-glutaminase co-activity. The more similar a biologic is to a human sequence, the less likely it would be immunogenic. In our work to date, we identified a mammalian L-asparaginase (referred to as gpASNas1) that is 70% identical to the human enzyme (as compared to the mere 25% identity of the bacterial enzymes), and we have increased that percentage identity to 85% by employing a genetic screen and structural information. In our proposed work here, we have identified a path that will increase this percent identity to >95%. As importantly, gpASNase1 is devoid of the toxicity-causing L-glutaminase activity, so as a drug, it will also lack those side effects that are caused by glutamine depletion. Critically, in a mouse xenograft model of human T-ALL and B-ALL, we observed a potent anti-cancer effect of these human-like L-asparaginase drugs, which serves to demonstrate that the L-glutaminase activity is not required for killing the cancer cells. Moreover, as compared to the L- glutaminase containing FDA drug, our L-asparaginase version without this co-activity has exhibited reduced toxicity. Thus, the L-asparaginase variant that will be developed by the proposed work will have a high impact on ALL therapy, especially for adults, and thus with special relevance for veterans. In addition to impacting ALL treatment, our vision is to expand the use of L-asparaginases to other malignancies. A main factor that currently prevents the expanded use of L-asparaginases (in addition to aforementioned side effects that will be largely reduced by our variants) is the lack of a method to identity patients who would most benefit from this drug. To remedy this deficiency and to promote personalizing medicine, we will first identify the factors that determine whether a cancer cell is sensitive or resistant to L- asparaginase, and then use this understanding to develop a predictive screen for L-asparaginase. Success in the proposed work will be transformative, as it will expand the use of L-asparaginases beyond ALL to other blood cancers, through the combination of a drug that is safer (by being less immunogenic and by lacking L-glutaminase co-activity) with a companion biomarker that can predict a patient's response to this drug.